Structural insights into the binding of cardiac glycosides to the digitalis receptor revealed by solid-state NMR

Abstract

Several biologically active derivatives of the cardiotonic steroid ouabain have been made containing NMR isotopes ( 13 C, 2 H, and 19 F) in the rhamnose sugar and steroid moieties, and examined at the digitalis receptor site of renal Na ؉ ͞K ؉ -ATPase by a combination of solid-state NMR methods. Deuterium NMR spectra of 2 H-labeled inhibitors revealed that the sugar group was only loosely associated with the binding site, whereas the steroid group was more constrained, probably because of hydrogen bonding to residues around the K ؉ -channel region. Crosspolarization magic-angle spinning NMR showed that chemical shifts of inhibitors 13 C-labeled in the sugar group moved downfield by 0.5 ppm after binding to the digitalis site, suggesting that the sugar was close to aromatic side groups. A 19 F, 13 C-rotational-echo double-resonance NMR strategy was used to determine the structure of an inhibitor in the digitalis receptor site, and it showed that the ouabain derivatives adopt a conformation in which the sugar extends out of the plane of the steroid ring system. The combined structural and dynamic information favors a model for inhibition in which the ouabain analogues lie across the surface of the Na ؉ ͞K ؉ -ATPase ␣-subunit with the sugar group facing away from the surface of the membrane but free to move into contact with one or more aromatic residues.

The non-gastric H,K-ATPase as a tool to study the ouabain-binding site in Na,K-ATPase

“…Keenan et al [28] have postulated a model in which the lactone ring is located close to the extracellular space and the rhamnose is embedded deeply into the membrane. The latter orientation does not fit with earlier nuclear magnetic resonance studies showing that the rhamnose has a high mobility [29].…”

Ouabain affinity determining residues lie close to the Na/K pump ion pathway

“…Mutagenesis has identified residues in the outermost segments of TM1, TM2, TM4, TM5, TM6, and TM7, and their connecting loops, that influence apparent affinity of ouabain binding or action (14, 17, 23, 34-37). Although evidence for direct interaction of these residues with cardiotonic steroids is lacking, the consensus interpretation pictures the steroid docking on the Na͞K pump's external surface, interacting with many of the identified amino acids (14,(38)(39)(40). Identification of the conserved Thr atop TM6 (34) and neighboring residues in the TM5-TM6 hairpin loop (35) as key determinants of ouabain apparent affinity led to the suggestion that cardiotonic steroids might act by preventing motion of the TM5-TM6 hairpin loop, thereby impeding ion passage to or from the deeper binding sites between TM4, TM5, and TM6 (35).…”

Detection of nucleotide binding to Na,K‐ATPase in an aqueous membrane suspension by 13C cross‐polarization magic‐angle spinning NMR spectroscopy

“…The Li-salt of [U- 13 C, 15 N]ATP is obtained from Silantes (Munich, Germany) and Li-salts of unlabelled ATP and ADP are obtained from Sigma-Aldrich (Denmark).…”

“…Hence it was necessary to develop a strategy that enables peaks from bound nucleotide to be detected under these transient conditions. It is shown that measurements of 13 C isotropic chemical shift values provide preliminary information about the nucleotide binding sites.…”

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Abstract Binding of uniformly 13 C labelled ATP to Na,KATPase was studied by 13 C cross-polarization magic-angle spinning (CP-MAS) NMR. In the presence of 30 mM Na + , and with sample-and time-averaging, NMR spectra obtained at 4°C exhibited several resonances for the bound nucleotide.

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